US11453882B2 - Heterologous biosynthesis of nodulisporic acid - Google Patents

Heterologous biosynthesis of nodulisporic acid Download PDF

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US11453882B2
US11453882B2 US16/651,065 US201816651065A US11453882B2 US 11453882 B2 US11453882 B2 US 11453882B2 US 201816651065 A US201816651065 A US 201816651065A US 11453882 B2 US11453882 B2 US 11453882B2
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Matthew Joseph Nicholson
Sarah Adeline Kessans
Emily Jane Parker
Leyla Yolanda Bustamante Rodriguez
David Barry Scott
Kyle Cornelius Van de Bittner
Craig John Van Dolleweerd
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Victoria Link Ltd
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    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)

Definitions

  • This invention generally relates to novel polypeptides that catalyze at least one biochemical reaction leading to the production of a nodulisporic acid (NA), polynucleotides encoding such polypeptides, methods of making such polypeptides and polynucleotides, and methods of using such polypeptides and polynucleotides to produce at least one NA by heterologous expression in a permissive host.
  • NA nodulisporic acid
  • Filamentous fungi produce a diverse repertoire of interesting and useful chemical compounds.
  • IDTs indole diterpenes
  • FIG. 1 are a group of notably bioactive quasi-paspaline-like IDTs produced by Hypoxylon pulicicidum , formerly classified as Nodulisporium sp. 7
  • Nodulisporic acid A (NAA) 10 is of particular significance because it exhibits highly potent insecticidal activity against blood-feeding arthropods while exhibiting no observable adverse effects on mammals. 5,8
  • NAs are especially difficult to biosynthesize from the natural producer, H. pulicicidum .
  • Reported NA biosynthesis methods require that H. pulicicidum be grown for 21 days in complete darkness in highly nutrient rich media.
  • Due to the difficulty of NAA 10 biosynthesis in H. pulicicidum obtaining useful quantities of NAA 10 using published fermentations methods is challenging, and production of commercial quantities of NAA 10 essentially unachievable. Accordingly, attempts have been made to chemically synthesize NAA 10 resulting in mechanisms for the synthesis of nodulisporic acid F (NAF) 5 a 10 and nodulisporic acid D 7 a, 11 but full synthesis of NAA 10 has not been achieved. 12 Consequently there is a need in the art for new methods of NAA 10 synthesis and/or biosynthesis that will provide useful quantities of NAA 10 .
  • NAF nodulisporic acid F
  • the invention relates to an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of NodW (SEQ ID NO:3), NodR (SEQ ID NO:6), NodX (SEQ ID NO:9), NodM (SEQ ID NO:12), NodB (SEQ ID NO:15), NodO (SEQ ID NO:18), NodJ (SEQ ID NO:21), NodC (SEQ ID NO:24), NodY1 (SEQ ID NO:27), NodD2 (SEQ ID NO:30), NodD1 (SEQ ID NO:33), NodY2 (SEQ ID NO:36), NodZ (SEQ ID NO:39), NodS (SEQ ID NO:50), and NodI (SEQ ID NO:56) or a functional variant or fragment thereof.
  • NodW SEQ ID NO:3
  • NodR SEQ ID NO:6
  • NodX SEQ ID NO:9
  • NodM SEQ
  • the invention relates to an isolated polynucleotide comprising at least 70% nucleic acid sequence identity to a nucleic acid sequence selected from the group consisting of nodW cDNA (SEQ ID NO:2), nodW genomic DNA (SEQ ID NO:1), nodR cDNA (SEQ ID NO:5), nodR genomic DNA (SEQ ID NO:4), nodX cDNA (SEQ ID NO:8), nodX genomic DNA (SEQ ID NO:7), nodM cDNA (SEQ ID NO:11), nodM genomic DNA (SEQ ID NO:10), nodB cDNA (SEQ ID NO:14), nodB genomic DNA (SEQ ID NO: 13), nodO cDNA (SEQ ID NO:17), nodO genomic DNA (SEQ ID NO:16), nodJ cDNA (SEQ ID NO:20), nodJ genomic DNA (SEQ ID NO:19), nodC cDNA (SEQ ID NO:23), nodC genomic DNA (SEQ ID NO:
  • the invention in another aspect relates to a transcription unit (TU) comprising at least one isolated polynucleotide according to the invention.
  • the invention in another aspect relates to a vector that encodes an isolated polypeptide according to the invention.
  • the invention in another aspect relates to a vector comprising an isolated nucleic acid sequence or a TU according to the invention.
  • the invention in another aspect relates to an isolated host cell comprising an isolated polypeptide, isolated polynucleotide, TU and/or vector according to the invention.
  • the invention in another aspect relates to a method of making at least one NA comprising heterologously expressing at least one polypeptide, isolated nucleic acid sequence, TU or vector according to the invention in an isolated host cell.
  • the invention relates to at least one NA made by a method of the invention.
  • the present invention relates to an isolated polypeptide or functional fragment or variant thereof from Hypoxylon spp. that catalyzes a biochemical reaction in the biosynthetic pathway leading from 3-geranylgeranyl indole (GGI) 2 to NAA 10 .
  • GGI 3-geranylgeranyl indole
  • the invention in another aspect relates to a method of making at least one Hypoxylon spp. polypeptide or functional variant or fragment thereof comprising heterologously expressing an isolated nucleic acid sequence or vector according to the invention in an isolated host cell.
  • the invention in another aspect relates to an isolated host cell that expresses at least one heterologous polypeptide that catalyzes the transformation of a substrate in the biosynthetic pathway leading from GGI 2 to the formation of NAA 10 .
  • FIG. 11 Overview of MIDAS Level-1 cloning.
  • A ggtctcgtgagacg (SEQ ID NO: 125); cgtctctagacc (SEO ID NO: 126); ccagagcactctgc (SEO ID NO: 127); gcagagatctgg (SEO ID NO: 128);
  • B cgtctcactcgggag (SEO ID NO: 129); aatgtgagacagagacg (SEO ID NO: 130); gcagagtgagccctc (SEO ID NO: 131); ttacactctgtctctgc (SEO ID NO: 132);
  • C ggtctcgggag (SEO ID NO: 133); aatgtgagacc (SEO ID NO: 134); ccagagccctc (SEO ID NO: 135); ttacactc
  • FIG. 14 Principle of MIDAS multigene assembly (level-3).
  • FIG. 15 Overview of MIDAS format.
  • genetic construct refers to a polynucleotide molecule, usually double-stranded DNA, which has been conjugated to another polynucleotide molecule.
  • a genetic construct is made by inserting a first polynucleotide molecule into a second polynucleotide molecule, for example by restriction/ligation as known in the art.
  • a genetic construct comprises a single polynucleotide module, at least two polynucleotide modules, or a series of multiple polynucleotide modules assembled into a single contiguous polynucleotide molecule (also referred to herein as a “multigene construct”), but not limited thereto.
  • transcription unit module refers to a polynucleotide comprising a sequence of nucleotides that encode a single RNA molecule, or parts thereof; or that encode a protein coding sequence (CDS), or parts thereof; or that encode sequence elements, or parts thereof, that control transcription of that RNA molecule; or that encode sequence elements or parts thereof that control translation of the CDS.
  • sequence elements may include, but are not limited to, promoters, untranslated regions (UTRs), terminators, polyadenylation signals, ribosome binding sites, transcriptional enhancers and translational enhancers.
  • marker means a nucleic acid sequence in a polynucleotide that encodes a selectable marker or scorable marker.
  • selectable marker refers to a TU, which when introduced into a cell, confers at least one trait on the cell that allows the cell to be selected based on the presence or absence of that trait. In one embodiment the cell is selected based on survival under conditions that kill cells not comprising the at least one selectable marker.
  • vector refers to any type of polynucleotide molecule that may be used to manipulate genetic material so that it can be amplified, replicated, manipulated, partially replicated, modified and/or expressed, but not limited thereto.
  • a vector may be used to transport a polynucleotide comprised in that vector into a cell or organism.
  • destination vector refers to a vector into which polynucleotide sequences of interest can be cloned.
  • the polynucleotide sequences are TUs and TUMs as described herein.
  • a destination vector is selected from the group consisting of plasmids, BACs, PACs, YACs, bacteriophage, phagemids, and cosmids.
  • a destination vector comprising a polynucleotide sequence of interest is an entry clone.
  • the entry clone can serve as a destination vector for receiving further polynucleotide sequences.
  • gene refers to gene the biologic unit of heredity, self-reproducing and located at a definite position (locus) on a particular chromosome.
  • particular chromosome is a eukaryotic or bacterial chromosome.
  • bacterial chromosome is used interchangeably herein with the term bacterial genome.
  • under conditions wherein the . . . enzyme is active” and “under conditions wherein the . . . enzymes are active”, and grammatical variations thereof when used in reference to enzyme activity mean that the enzyme will perform it's expected function; e.g., a restriction endonuclease will cleave a nucleic acid at an appropriate restriction site, and a DNA ligase will covalently join two polynucleotides together.
  • a wild type polynucleotide sequence is a naturally occurring polynucleotide sequence, but not limited thereto.
  • a naturally occurring polynucleotide sequence also refers to variant polynucleotide sequences as found in nature that differ from wild type. For example, allelic variants and naturally occurring recombinant polynucleotide sequences due to hybridization or horizontal gene transfer, but not limited thereto.
  • wild type when used herein with reference to a polypeptide refers to a naturally occurring, non-mutant form of a polypeptide.
  • a wild type polypeptide is a polypeptide that is capable of being expressed from a wild type polynucleotide.
  • “Introduced Homologous” as used herein with reference to polynucleotide or polypeptide in a host organism means that the polynucleotide or polypeptide is a native and naturally-occurring polynucleotide or polynucleotide within that host organism that has been introduced into the organism by experimental techniques.
  • a introduced homologous polynucleotide may be operably linked to a homologous or heterologous regulatory element so that a homologous polypeptide may be expressed from a TU, genetic element or vector comprising the homologous polynucleotide as described herein.
  • Heterologous as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is not a native and naturally-occurring polynucleotide regulatory element.
  • a heterologous polynucleotide regulatory element is not normally associated with the CDS to which it is operably linked.
  • a heterologous regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a, vector, genetic construct or expression cassette according to the invention.
  • Such promoters may include promoters normally associated with other genes, ORFs or coding regions, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell.
  • Heterologous as used herein with reference to a polynucleotide or polypeptide in a host organism means a polynucleotide or polypeptide that is not a native and naturally-occurring polynucleotide or polypeptide in that host organism.
  • a heterologous polynucleotide may be operably linked to a heterologous or homologous regulatory element so that a heterologous polypeptide may be expressed from a TU, genetic element or vector comprising the heterologous polynucleotide as described herein.
  • a “biochemical reaction in the biosynthetic pathway leading from GGI 2 to NAA 10 ” means one of the specific reactions catalyzed by one of the specific enzymes involved in transforming the substrate molecule GGI 2 through the following intermediates: mono-expoxidized GGI 3 a , emindole SB 4 a , NAF 5 a , NAE 6 a , NAD 7 a , NAC 8 , NAB 9 , to NAA 10 , and does not include similar enzymes within a host cell that may have similar functions but that do not act on the particular named intermediates above.
  • isolated when used herein in reference to a cell or host cell describes to a cell or host cell that has been obtained or removed from an organism or from its natural environment and is subsequently maintained in a laboratory environment as known in the art.
  • the term encompasses single cells, per se, as well as cells or host cells comprised in a cell culture and can include a single cell or single host cell.
  • variants refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities that are the same or similar to those of a corresponding wild type molecule; i.e., the parent polypeptides or polynucleotides.
  • variants with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.
  • Polynucleotide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.
  • Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention.
  • a skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
  • variant polypeptide sequences preferably exhibit at least 35%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%,
  • a variant polypeptide includes a polypeptide wherein the amino acid sequence differs from a polypeptide herein by one or more conservative amino acid or non-conservative substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide.
  • variants include peptides with modifications which influence peptide stability.
  • Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence.
  • analogs that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta or gamma amino acids and cyclic analogs.
  • the cloned CDSs were placed under the control of heterologous promoter (ProUTR) and transcriptional terminator (UTRterm) modules to generate full-length TUs (Table 10), which were then used to generate the multi-gene plasmids (Table 11).
  • the inventors used a repertoire of P. paxilli knockout strains (Table 7) to carry out functional complementations and pathway reconstitution to determine the functions of genes in the NOD cluster. Following transformation of P.
  • NodM SEQ ID NO:12
  • PaxM SEQ ID NO:47
  • NodB SEQ ID NO:15
  • H. pulicicidum acts as the IDT cyclase that cyclizes the monoepoxidized-GGI product 3 a to form emindole SB 4 a ( FIG. 3 , trace iii.b, FIG.
  • the isolated polypeptide comprising NodJ (SEQ ID NO:21) or a functional variant or fragment thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity.
  • the isolated polypeptide comprising NodC (SEQ ID NO:24) or a functional variant or fragment thereof has transferase activity, preferably GGT activity.
  • the isolated polypeptide comprising NodY1 (SEQ ID NO:27) or a functional variant or fragment thereof has oxygenase activity, preferably FAD-dependent oxygenase activity.
  • the isolated polypeptide comprising NodY2 (SEQ ID NO:36) or a functional variant or fragment thereof has oxygenase activity, preferably FAD-dependent oxygenase activity.
  • the isolated polynucleotide encodes a polypeptide comprising NodO (SEQ ID NO:18) or a functional variant or fragment thereof having oxygenase activity, preferably FAD-dependent oxygenase activity.
  • the fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized as known in the art. Primers and primer pairs which allow amplification of polynucleotides of the invention, also form a further aspect of this invention.
  • Variants may be identified by the methods described. Variant polynucleotides may be identified using PCR-based methods as known in the art.
  • the invention in another aspect relates to a TU comprising at least one isolated polynucleotide as described herein.
  • the TU is comprised in vector, preferably an expression vector.
  • the vector is selected from the group consisting of plasmids, BACs, (PACs), YACs, bacteriophage, phagemids, and cosmids.
  • the vector is a plasmid.
  • the vector is a component in a cloning system.
  • the cloning system is useful for making a gene construct comprising at least one TU.
  • the gene construct is a multigene construct comprising at least two TUs.
  • the multigene construct comprises at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten TUs.
  • the TUs described herein may comprise one or more of the disclosed polynucleotide sequences and/or polynucleotides encoding the disclosed polypeptides, of the invention.
  • the TU can constructed to drive expression of at least one polypeptide involved in the biosynthesis of NAA 10 , either in vitro or in vivo.
  • the TU comprises a polynucleotide of the invention operatively linked to 5′ or 3′ untranslated regulatory sequences.
  • the design of a particular TU will depend on various factors including the host cells in which the operatively linked polynucleotide is to be expressed and the desired level of polynucleotide expression.
  • the selection of various promoters, enhancers and/or other genetic elements for a TU will depend on various factors including the host cells and expression levels discussed above.
  • the TU comprises a homologous promoter operatively linked to a polynucleotide of the invention.
  • the expression cassette comprises a heterologous promoter operatively linked to a polynucleotide of the invention.
  • the homologous or heterologous promoter is an inducible, repressible or regulatable promoter.
  • a suitable promoter may be chosen and used under the appropriate conditions to direct high-level expression of a polynucleotide of the invention. Many such elements are described in the literature and are available through commercial suppliers.
  • promoters useful in the expression cassettes can be any suitable eukaryotic or prokaryotic promoter.
  • the eukaryotic promoter can be a eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).
  • poly I eukaryotic RNA polymerase I
  • poly II RNA polymerase II
  • poly III RNA polymerase III
  • Expression levels of an operably linked polynucleotide in a particular cell type will be determined by the nearby presence (or absence) of specific gene regulatory sequences (e.g., enhancers, silencers and the like).
  • Any suitable promoter/enhancer combination see: Eukaryotic Promoter Data Base EPDB can be used to drive expression of a polynucleotide of the invention.
  • Prokaryotic promoters useful in expression cassettes include constitutive promoters as known in the art (such as the int promoter of bacteriophage lamda and the bla promoter of the beta-lactamase gene sequence of pBR322) and regulatable promoters (such as lacZ, recA and gal).
  • constitutive promoters such as the int promoter of bacteriophage lamda and the bla promoter of the beta-lactamase gene sequence of pBR322
  • regulatable promoters such as lacZ, recA and gal.
  • a ribosome binding site upstream of the CDS may also be required for expression.
  • Enhancers useful in a TU include SV40 enhancer, cytomegalovirus early promoter enhancer, globin, albumin, insulin and the like.
  • the TU is comprised in a multigene construct comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine and/or at least 10 polynucleotides encoding polypeptides according to the invention.
  • the present invention relates to an isolated polynucleotide encoding at least one polypeptide from Hypoxylon spp. that catalyzes a biochemical reaction in the biosynthetic pathway leading from GGI 2 to NAA 10 .
  • the isolated polypeptide is an oxygenase, preferably a cytochrome P450 oxygenase or a FAD-dependent oxygenase.
  • the cytochrome P450 oxygenase is NodW (SEQ ID NO:3), NodR (SEQ ID NO:6), NodX (SEQ ID NO:9), NodJ (SEQ ID NO:21), or NodZ (SEQ ID NO:39).
  • the FAD dependent oxygenase is NodM (SEQ ID NO:12), NodO (SEQ ID NO:18), NodY1 (SEQ ID NO:27), or NodY2 (SEQ ID NO:36).
  • the isolated polypeptide catalyzes a biochemical reaction in the biosynthetic pathway leading from GGI 2 to NAF 5 a .
  • the isolated polypeptide is a GGT, a FAD-dependent oxygenase, an IDT cyclase, or a cytochrome P450 oxygenase.
  • the GGT is NodC (SEQ ID NO:24).
  • the FAD-dependent oxygenase is NodM (SEQ ID NO:12).
  • the IDT cyclase is NodB (SEQ ID NO:15).
  • the cytochrome P450 oxygenase is NodW (SEQ ID NO:3).
  • the isolated polypeptide or functional variant or fragment thereof is encoded by a nucleic acid according to the invention.
  • the at least one Hypoxylon spp. polypeptide is a polypeptide comprising an amino acid sequence of SEQ ID NO: NodW (SEQ ID NO:3) or a functional variant or fragment thereof.
  • the polypeptide consists essentially or consists of SEQ ID NO: NodW (SEQ ID NO:3).
  • the isolated host cell comprises fungal mycelia of the genus Penicillium , preferably P. paxilli.
  • the substrate that is transformed is NAB 9 and the transformation is an oxidation.
  • the at least one polypeptide is a polypeptide involved in the biosynthetic pathway leading to NAA 10 as defined herein for any other aspect of the invention.
  • the invention in another aspect relates to a method of making NAA 10 comprising expressing at least one heterologous nucleic acid sequence in Hypoxylon pulicicidum , wherein the at least one heterologous nucleic acid sequence encodes an enzyme in a biosynthetic pathway leading to NAA 10 .
  • the recipient plasmid contains a marker gene (typically the lacZa gene for blue/white screening) flanked by two divergently oriented recognition sites for a Type IIS enzyme; these elements, collectively called the ‘Golden Gate cloning cassette’, are replaced by the insert during the assembly reaction.
  • a marker gene typically the lacZa gene for blue/white screening
  • two divergently oriented recognition sites for a Type IIS enzyme these elements, collectively called the ‘Golden Gate cloning cassette’, are replaced by the insert during the assembly reaction.
  • the Golden Gate cloning cassette present in pML1 consists of two divergent BsmBI sites flanking a lacZa scoreable marker: 5′-[CTCG]BsmBI-lacZa-BsmBI[AGAC]-3′ ( FIG. 11A ).
  • a CDS module would be flanked by AATG and GCTT (i.e., 5′-A ATG -CDS-GCTT-3′), while a UTRterm module (consisting of a 3′UTR and a 3′ non-transcribed region, including the polyadenylation signal) would have the form 5′-GCTT-UTRterm-CGCT-3′.
  • the module-specific bases GGAG, located at the 5′ end of ProUTR modules, and CGCT, at the 3′ end of UTRterm modules, are compatible with the overhangs generated by BsaI digestion of the pML2 destination vectors, and these bases therefore define the outermost cloning boundaries of a Level-2 assembly.
  • Level-2 Level-2
  • pML2 Level-2 destination vectors into which a TU can be assembled, the choice of which depends on the desired configuration of TUs in the multigene plasmid produced at Level-3, namely: (i) the desired order in which TUs are added to the multigene assembly, (ii) the desired direction in which the multigene plasmid is assembled and (iii) the desired orientation of each TU in the multigene plasmid.
  • the pML2 vectors are distinguished from one another by the arrangement of specific sequence features that are central to the operation of MIDAS. These sequence features, collectively called the MIDAS cassette ( FIG. 13 ), define the Level-2 assembly of TUs and govern the assembly of multigene constructs produced at Level-3.
  • PCR-amplified modules were purified using spin-column protocols and cloned into the MIDAS Level-1 plasmid, pML1, by BsmBI-mediated Golden Gate assembly.
  • 1-2 ⁇ L (approximately 50-200 ng) of pML1 plasmid DNA from a miniprep was mixed with 1-2 ⁇ L of each purified PCR fragment, 1 ⁇ L of BsmBI (20 U/ ⁇ L), 1 ⁇ L of T4 DNA Ligase (20 U/ ⁇ L) and 2 ⁇ L of 10 ⁇ T4 DNA Ligase buffer in a total reaction volume of 20 ⁇ L. Reactions were incubated at 37° C.
  • Mycelia were weighed, resuspended in 10 mL of filter-sterilized Lysing Enzymes solution (prepared by resuspending Lysing Enzymes from Trichoderma harzianum (Sigma) at 10 mg/mL in OM buffer) per gram of mycelia, and incubated for 16 hours at 30° C. with shaking at 80 rpm. Protoplasts were filtered through a sterile nappy liner into a 250 mL Erlenmeyer flask.
  • filter-sterilized Lysing Enzymes solution prepared by resuspending Lysing Enzymes from Trichoderma harzianum (Sigma) at 10 mg/mL in OM buffer
  • the white layer of protoplasts that formed between the OM and ST buffers in each tube was transferred (in 2 mL aliquots) into sterile 15 mL centrifuge tubes, gently washed by pipette resuspension in 5 mL of STC buffer (1 M sorbitol, 50 mM Tris-HCl at pH 8.0, and 50 mM CaCl 2 ) and centrifuged at 2600 ⁇ g for 5 minutes at 4° C. The supernatant was decanted off and pelleted protoplasts from multiple tubes were combined by resuspension in 5 mL aliquots of STC buffer.
  • the STC buffer wash was repeated three times until protoplasts were pooled into a single 15 mL centrifuge tube.
  • the final protoplasts pellet was resuspended in 500 ⁇ L of STC buffer and protoplast concentration was estimated with a hemocytometer.
  • the protoplast stock was diluted to give a final concentration of 1.25 ⁇ 10 8 protoplasts per mL of STC buffer. Aliquots of protoplasts (100 ⁇ L) were used immediately for fungal transformations and excess protoplasts were preserved in 8% PEG solution (80 ⁇ L of protoplasts were added to 20 ⁇ L of 40% (w/v) PEG 4000 in STC buffer) in 1.7 mL micro-centrifuge tubes and stored at ⁇ 80° C.
  • the transformation mixture was incubated on ice for a further 15-20 minutes, transferred to 17.5 mL of 0.8% RGA medium (prewarmed to 50° C.) in sterile 50 mL tubes, mixed by inversion, and 3.5 mL aliquots were dispensed onto 1.5% RGA plates. Following overnight incubation at 25° C., 5 mL of 0.8% RGA (containing sufficient geneticin to achieve a final concentration of 150 ⁇ g per mL of solid media) was overlaid onto each plate. Plates were incubated for a further 4 days at 25° C. and spores were picked from individual colonies and streaked onto CDYE agar plates supplemented with 150 ⁇ g/mL geneticin.
  • Level-2 entry plasmids produced are shown in bold. This are described by the CDS they contain and TU orientation, determined by the pML2 destination vector, is shown by the arrowhead ( for forward (F) destination vector and for reverse (R) destination vector) in the Level-2 description.
  • Level-3 multigene assemblies are constructed by alternating Golden Gate cloning reactions using TUs assembled in “White” and “Blue” pML2 vectors.
  • Destination Golden Gate Step Level-2 entry clone plasmid reaction Product plasmid Screen 1 TU1 in a White pML2 vector pML3 AarI-mediated pML3:TU1 White colonies 2 TU2 in a Blue pML2 vector pML3:TU1 BsmBI-mediated pML3:TU1:TU2 Blue colonies 3 TU3 in a White pML2 vector pML3:TU1:TU2 AarI-mediated pML3:TU1:TU2:TU3 White colonies 4 TU4 in a Blue pML2 vector pML3:TU1:TU2:TU3 BsmBI-mediated pML3:TU1:TU2:TU3:TU4 Blue colonies
  • the table shows the cloning steps used to produce a hypothetical multigene construct containing four TUs, with

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